WO2012064096A2 - Novel strain of bacillus subtilis, and use thereof for preventing root rot disease in plants - Google Patents

Abstract

The present invention relates to a novel strain of Bacillus subtilis, and more particularly, to the B-5604 strain of Bacillus subtilis, which inhibits the growth of root rot pathogens, to a composition containing said strain or the culture medium thereof for controlling root rot pathogens, to a method for controlling ginseng root rot pathogens using said composition, and to a method for inhibiting the growth of Cylindrocarpon destructans or Fusarium solani using said strain. The microorganisms having antagonistic activity developed according to the present invention are not harmful to water and soil, and thus does not cause environmental pollution, and particularly, the microorganisms having antagonistic activity according to the present invention may not only reduce root rot pathogens in farmland in which ginseng is repeatedly cultivated or recultivated, but also alleviate root rot disease in ginseng being cultivated, thus significantly contributing to ginseng cultivation.

Description

Novel Bacillus subtilis strains and their use for preventing plant root rot

The present invention relates to a novel Bacillus subtilis strain, more specifically Bacillus subtilis strain B-5604, which inhibits the growth of root rot pathogens, the composition for the control of the root rot pathogens comprising the strain or its culture solution, and It relates to a method for controlling the ginseng root rot pathogens using the composition and to inhibit the growth of Sirindrocarpon destructans or Fusarium solani using the strain.

Studies on the root rot of plants before 1992 were mainly due to the study of a wide range of root rot caused by the compound root rot and the ginseng root rot pathogen Cylindrocarpon destructans (Zinssm.) Scholten. Although actual research on root rot has been conducted since 1992. However, there are still few studies on this pathogen. Worldwide, the pathogens are reported to be mainly caused by trees or bulbs such as daffodils, cyclamen, alfalfa and ginseng (Table 1).

Table 1

Ginseng root rot disease control research has been tried by various researchers. However, the mechanism of pathogenesis of root rot disease caused by syringrocapon destructans and the physiology and ecological basic research of pathogens are insufficient. There is no way to control it. In addition, the root rot was identified as the root rot pathogen in the soil, but after the onset of the effective control means, the root rot caused after ginseng transplantation only prevented the spread of the surrounding environment. In ginseng cultivation, harvesting ginseng and cultivating other crops for 7-10 years, then cultivating ginseng again, and cultivating ginseng, harvesting ginseng, and cultivating ginseng again for 1-2 years without growing other crops. It is tilling. Ginseng is a representative crop that causes obstacles caused by crops. In the main ginseng producing area, there is an absolute shortage of cropland due to the increase of the required growing area. . However, the production and quality of ginseng deteriorated due to the lack of adaptation due to the increase of production cost, neglect of management and the change of weather and soil conditions in new areas. Sometimes. When ginseng is re-cultivated or re-created within 4-5 years after harvesting ginseng, root rot occurs when the ginseng package becomes dark brown and leaves become prematurely reddened.

Ginseng cultivators recognize that the initial level of root rot pathogens in cropland is not so high that the initial root rot disease occurs at least 50% when harvested for at least 6 years. In general, it is thought to be part of the 50% that is lost during cultivation. However, in the field of serial disturbance, initial colonization rate of root rot disease reached 30% and severe cases reached 80% within 1-2 years after transplantation.

The symptom of serial disturbances is that the roots rot to dark brown, and the leaves dry out early, causing the roots to rot.Cylindrocarpon destructans (Zinssm.) Scholten was confirmed to be pathogenic to ginseng, and the physiological characteristics of the pathogen and the availability and secretion of amino acids in the culture were studied. Initially, this bacteria was used (C. radicicola) And was renamed as Syringrocarpon Destructans by Scholten. Differences in mycelial growth and toxin production in the growth medium of cyndrocarpon destructans and the effects of temperature on the growth of these bacteria have been reported. Growth differences due to various factors, such as the culture temperature, light, the pH of the medium, the type of medium has been reported. Subsequently, reports related to growth characteristics were confirmed by the re-separation of the syringdrocarpon destructans as root rot pathogens related to ginseng's softening disorders. For the development of selective medium, the growth characteristics of the culture conditions such as temperature, pH and the addition of antimicrobial substances have been reported.

Pathogens related to root rot in ginseng cultivationPhytophthora cactorum),Cylindro car phone destruction tanks (Cylindrocarpon destructans),Fusarium Solani (Fusarium solani) And the like have been reported. Among these pathogens, damage caused by Cylindrocarpon destructans, which is known as a causative agent of serial disturbance and forms a thick film spore, causes disease due to soil infectiousness. And with the isolation and pathogenicity of Cylindrocarpon distractans from the typical black lesions of the disease, interest in the pathogens has increased. It is reported that the pathogens need attention in the root rot caused by the Syringrocarpon distractans in not only the field but also the field.

In the meantime, as a method of reducing the density of soil pathogens present in the soil, ginseng is desalted in cultivated soil, and a method of using a soil fumigant has been known, but there are disadvantages in using the method. After the organic mercury fumigant was treated in the soil harvested with ginseng in autumn and the density change of soil microorganisms was examined over time, the bacteria with high growth rate recovered to the level after 20 days of treatment, but the density of fungi or actinomycetes with slow growth rate The rapid decrease immediately after treatment, the microbial density returned to the normal level at the time of ginseng transplanting in the following spring, so the control effect of harmful fungi in soil was not significant. Root rot pathogens are inoculated at 30cm from the soil surface and ginseng is transplanted, but root rot disease occurs when the root rot pathogens are inoculated at 50 cm or less. Although a little expected, if the roots rot by root rot pathogens during ginseng cultivation, in the state that the ginseng is being cultivated, it is difficult to use the fumigant because it is impossible.

Thus, in order to prevent root rot, which is the biggest problem in ginseng cultivation, many researchers at home and abroad have tried to control with various cultivation methods and chemical synthetic pesticides, but it is difficult to use due to the low control value of selected pesticides and some growers. Is indiscriminately polluting water quality and soil as well as raw ginseng. In addition, red ginseng products are also subject to restrictions on pesticides used by importing countries in foreign countries. Therefore, care should be taken in the use of pesticides remaining in pesticides permitted in Korea even if they are licensed in Korea. Nowadays, the importance of the development of environmentally friendly low-toxic pesticides with a definite mechanism of action due to concern about damage caused by organically synthesized pesticides and various environmental pollutions is widely recognized.

The present inventors have made diligent efforts to find new fungicides that can control environmentally-friendly and low-toxic root-mixing diseases, and as a result, ginseng root rot-inducing microorganisms from environmentally-friendly microbial resources for controlling root rot caused during the planting and re-production of ginseng. Newly developed antagonistic microorganisms to control the present invention was completed. The active antagonistic microorganisms developed in the present invention have no environmental pollution because they are friendly to water quality and soil, and in particular, the active antagonist microorganisms are innovative for ginseng cultivation because they improve the root rot disease of the ginseng under cultivation or re-cultivation of ginseng. It is expected to contribute.

One object of the present invention is to provide a novel Bacillus subtilis strain B-5604 (Accession Number: KCTC 11758BP).

Another object of the present invention is to provide a composition for controlling root rot pathogens comprising the strain or its culture as an active ingredient.

Another object of the present invention to provide a method for controlling the ginseng root rot pathogens using the composition.

Another object of the present invention is to use the strain strains of Syringrocarpon destructans (Cylindrocarpon destructans) or Fusarium Solani (Fusarium solaniIt is to provide a method of suppressing the growth of).

Still another object of the present invention is to provide a method for controlling root rot pathogens of the strain or composition.

The antagonistic microorganisms developed in the present invention have a new mechanism of action by making a search system that can search for active substances for bactericidal activity of the new mechanism of action by identifying that there is a bactericidal resistance mechanism specific to ginseng root rot disease. It may be used as an active microorganism capable of overcoming the mechanism of bactericidal resistance. In addition, by effectively inhibiting the growth of the root rot causing bacteria of ginseng, it does not cause environmental pollution to ginseng cultivation and reduces the management cost and environmental pollution of domestic ginseng cultivating farmers by supplying eco-friendly ginseng root rot control or pesticide that is not toxic to human body. It will also contribute to the stable supply and export of high-quality ginseng products with high competitiveness. And because Fusarium Solani strain is known to cause problems not only in American ginseng but also Japanese ginseng The secured active microorganisms are expected to enhance the national competitiveness and contribute to the vitalization of the national economy.

1 is a diagram showing an antagonistic strain showing inhibitory activity on Cylindrocarpon destructans .

Figure 2 is a diagram showing the sequence of the gene rDNA encoding the 16S rRNA of Bacillus subtilis B-5604 of the present invention (SEQ ID NO: 1).

Figure 3 is an electron micrograph of the antagonist microorganism of the present invention.

Figure 4 is a diagram showing the results of plant root rot disease prevention of antagonism microorganisms.

5 is a view showing the results according to the treatment of antagonism microorganisms in plants.

In one embodiment, the present invention provides a novel Bacillus subtilis strain B-5604 (Accession Number: KCTC 11758BP).

The strain is characterized in that it inhibits the growth of root rot pathogens, preferably can inhibit the growth of ginseng root rot pathogens, and thereby can control the ginseng root rot disease that has emerged as a major problem in cultivation of ginseng. have.

In the present invention, the term "ginseng root rot" is a soil plant disease that occurs more than 50% during cultivation of ginseng roots is a disease in which the roots rot to dark brown and leaves become prematurely reddened. To date, there is a lack of reliable prevention and control methods due to insufficient pathogenesis of ginseng root rot, physiology and ecological basic research of pathogens. As a result of the present inventors' efforts to develop a new prevention or control method, the bacillus subtilis strain B-5604, a novel strain developed by the present inventors, is a strain of ginseng root rot pathogen, which is a kind of ginseng root rot pathogen. (Cylindrocarpon destructans) or Fusarium Solani (Fusarium solaniIt was first identified that ginseng root rot can be prevented by suppressing the growth of the.

The strain of the present invention is a strain having an antagonistic ability to inhibit the growth of the root rot causing strains, preferably can suppress the growth of the strain causing ginseng root rot. In the present invention, "ginseng root rot pathogen" includes a strain that can cause ginseng root rot, without limitation, for example, phytopsorra cockroach (Phytophthora cactorum),Cylindro car phone destruction tanks (Cylindrocarpon destructans),Fusarium Solani (Fusarium solaniAnd the like, and preferably, the pathogen which can inhibit growth of the strain of the present invention is Sirandrocarpon destructans. (Cylindrocarpon destructans) Or Fusarium Solani (Fusarium solaniCan be). According to a specific embodiment of the present invention, it was confirmed that the strain can inhibit the growth of the Syringrocarpon distractans or Fusarium solani (see Examples 1 and 4).

In the present invention, the term "cylindrocaphone destructives" (Cylindrocarpondestructans) "is a destructans (scavenger) squalton (Cylindrocarpon destructans (Zinssm.) Scholten) can be used interchangeably. The pathogen is known as a causative agent of ginseng consecutive disorders, it has been found to form a thick film spores, causing the disease infectious soil. In addition, the strain was isolated from the typical black lesion tissue of the root rot disease, pathogenicity was confirmed, and if the growth of the pathogen is inhibited, ginseng root rot disease can be controlled soon.

As used herein, the term " Fusarium solani " is a bacterium known as a pathogen of ginseng root rot, and when infected with the fungus, the surface of the ginseng root becomes light brown or reddish brown and soft to the root. do.

The present inventors isolated the mutation-induced antagonistic strains by exposure to ultraviolet rays while searching for strains showing inhibitory activity against the Fusarium solanie strain and the ginseng root rot strain of ginseng root rot. Were separated from soil samples (FIG. 1).

The sequence of the gene (rDNA) encoding the 16S rRNA of the strain provided by the present invention is set forth in SEQ ID NO: 1 (FIG. 2). Compared to the rDNA sequence of the known strain, the sequence of the rDNA set forth in SEQ ID NO: 1 shows 99% similarity to the known Bacillus subtilis strain AB017589 and 99% identity to another known Bacillus subtilis strain AB017590 Indicated. The size of the strains provided by the present invention was on average 0.6-0.7 X 1.5-1.6 μm, slightly smaller than the general Bacillus subtilis size of 1.2-2.5 μm, with one flagella, and its length was approximately 7.5 μm. Was about 5 times (Fig. 3). As a result of comparing the classification position of the strain, it was classified as Bacillus subtilis, and the strain of the present invention was named "Bacillus subtilis B-5604" based on the characteristics of fatty acid components and ratios, cell appearance, and the like. On September 13, 2010, it was deposited with the accession number KCTC 11758BP at the Korea Research Institute of Bioscience and Biotechnology, Korea Institute of Bioscience and Biotechnology, 111, Gwahak-ro, Yuseong-gu, Daejeon, Korea.

According to one embodiment of the present invention, it was confirmed that the strain of the present invention inhibits the growth of Fusarium solani and Sirandrocarpon destructans (see Example 4). In addition, to investigate whether the strain of the present invention has a ginseng root rot inhibitory effect, three quarters of the ginseng roots are covered with soil containing the root rot pathogen, and the above is adsorbed to the vermiculite, etc. As a result of covering the dried soil, the soil part where root rot pathogen was present was rotted as it was, but the part grown at the drug attachment part adsorbed to the vermiculite such as the root rot antagonist microorganism was improved and the root rot disease was improved. It confirmed that the state was favorable (FIG. 4). In addition, a test plot covering the ginseng planted by mixing the vermiculite containing root rot pathogens with soil containing Root rot pathogens after adsorbing control microorganisms of root rot pathogens to the same amount of vermiculite as a control. As a result of comparison, the soil in which the root rot pathogens existed was much rotten, but in the group treated with the antagonist microorganism of the present invention, it was confirmed that the root rot disease was improved and the growth condition was good (FIG. 5). These results suggest that the strain developed in the present invention can effectively control ginseng root rot. Therefore, the novel Bacillus subtilis strain B-5604 of the present invention has excellent antagonistic ability against ginseng root rot pathogen, and can be used as an environment-friendly ginseng root rot pathogen control microorganism because there is no environmental pollution and human toxicity. have.

As another aspect, the present invention provides a composition for the control of root rot pathogens comprising Bacillus subtilis strain B-5604 (Accession Number: KCTC 11758BP) or a culture thereof as an active ingredient.

Root rot pathogens are as described above.

The compositions of the present invention may be formulated in the form of hydrates, granules or encapsulations for the purpose of stable formulation of microorganisms.

In the composition of the present invention, the microorganism or the culture medium may be supplied as contained in the composition for controlling ginseng root rot pathogen, or may be stored separately for long-term storage and mixed before use. When the microorganisms are supplied separately for long term storage, they may be stored at -70 ° C. or lower in glycerol stock solution or may be used by freeze-drying at -20 to -80 ° C.

The hydrating agent of the present invention may be prepared by drying and grinding a solid medium inoculated with microorganisms, followed by mixing by adding a surfactant and an extender / nutrient. In the above surfactants, polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene Group consisting of alkyl aryl phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkylone alkyl phenyl ethers, polyoxyethylene nonyl phenyl ethers, sodium sulfonate naphthalene formaldehyde, Triton 100 and Tween 80 One or two or more selected from among them may be used, and one or two or more selected from the group consisting of soy flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose and starch may be used as extenders and nutrients.

In addition, the granules of the present invention may be prepared by drying and grinding a solid medium inoculated with microorganisms and then adding a surfactant, an extender / nutrient and a disintegrant. In the above surfactants, polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene Group consisting of alkyl aryl phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkylone alkyl phenyl ethers, polyoxyethylene nonyl phenyl ethers, sodium sulfonate naphthalene formaldehyde, triton 100 and twin 80 One or two or more selected from the group consisting of one or two or more selected from the group consisting of soy flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose and starch, and bentonite as a disintegrant. (bentonite), talc, dialite, One or more than one selected from the group consisting of kaolin and calcium carbonate can be used.

In addition, the granules of the present invention may include one or two or more selected from the group consisting of surface active agents, inert carriers, preservatives, wetting agents, feed promoters, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protectors, buffers and flow agents for microorganisms. It can be prepared by adding additional.

The composition can be applied to the root of the plant or the soil around it to control the root rot pathogen. More preferably, it can be applied to the roots of or around the ginseng to control the ginseng root rot pathogens. The method of applying the composition may be directly spreading, spraying, soaking the culture or strain of the strain, or scattering or cultivating the strain culture or the strain by adsorbing on vermiculite, diatomaceous earth, perlite, or tilling. In the management of the planned site, a method of mixing the strain culture medium or the adsorbed strains during soil plowing can be used. In addition, known pesticide administration methods, microbial application methods and the like can be used.

Application of the composition is sprayed by concentrating Bacillus subtilis B-5604 in the roots or adjacent soil of ginseng, the effective amount of microorganisms is 1 X 10 ⁷ to 5 X 10 ⁷ May be sparged, preferably 4 × 10 ⁷ birds.

In addition, the application of microorganisms can be achieved by spraying the roots of ginseng with a solution in which the effective amount of microorganisms contained in the composition is diluted at a concentration of 1 X 10 ⁶ to 1 X 10 ⁹ microorganisms per ml (ml), or per ml (ml) Ginseng may be immersed in a solution diluted to a concentration of 1 X 10 ⁶ to 1 X 10 ⁹ microorganisms for 1 to 2 hours.

As another aspect, the present invention is a method for controlling ginseng root rot pathogens using the composition for the control of root rot pathogens comprising Bacillus subtilis strain B-5604 (Accession Number: KCTC 11758BP) or a culture thereof as an active ingredient. To provide.

The method of controlling the ginseng root rot pathogens can be controlled by spraying or immersing the composition in the root or the soil adjacent to the ginseng, or immersed, the details are as described above.

In addition, the composition can inhibit the growth of ginseng root rot pathogens to control the ginseng root rot pathogens, the description of the ginseng root rot pathogens is as described above.

In another embodiment, the present invention provides a Cylindrocarpon distractans using Bacillus subtilis strain B-5604 (Accession Number: KCTC 11758BP). (Cylindrocarpon destructans) or Fusarium Solani (Fusarium solaniIt provides a method of suppressing the growth of).

The strains, strains that are subject to growth inhibition and growth methods are as described above.

In another embodiment, the present invention comprises Bacillus subtilis strain B-5604 (Accession No .: KCTC 11758BP), or Bacillus subtilis strain B-5604 (Accession No .: KCTC 11758BP) or its culture solution as an active ingredient. Provided is a method for controlling root rot pathogens of the composition for root rot pathogen control.

Control uses may be carried out by controlling the root rot pathogens by spraying or soaking the strain or composition concentrated on the root of the plant or the soil adjacent thereto, and the details are as described above. Preferably the plant may be ginseng, thereby controlling the ginseng root rot pathogens.

In addition, the strain or composition may control the growth of the root rot pathogens by inhibiting the growth of the root rot pathogens, the description of the root rot pathogens as described above.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Selection of Antagonist Microorganisms

The present inventors searched for antagonistic microorganisms among microorganisms that can also grow in the inhibitory activity produced by the root rot pathogens in the soil samples of Chungcheongnam-do in order to search for antagonistic microorganisms that inhibit the growth of root rot pathogens of ginseng. Investigation of antagonistic microorganisms that inhibit the growth of root rot pathogens of ginseng is carried out by diluting the soil sample in distilled water stepwise to spread the inhibitory substances produced by the root rot pathogens on a separation medium, and then selecting and separating candidate strains. Was performed.

The microorganism separation medium used by the present inventors (yeast extract 0.1%, polypeptone 0.5%, potassium monohydrogen phosphate 0.05%, potassium diphosphate 0.05%, magnesium sulfate seven times crystallized water 0.02%, agar 1.5%, hydrogen ion concentration 7.2) After sterilization, the temperature was lowered to about 50 ° C, and a solution of colistin dissolved in distilled water at a concentration of 5 mg / L as an added antibiotic and 10 mg / of nalidixic acid was added to suppress growth of general bacteria. 1 ml per 1 liter of the sterilized separation medium in which the solution dissolved in methanol at a concentration of l was mixed well and then a solidified separation medium was used. Diluting the soil sample in the prepared separation medium in stages and spreading and incubating at 28 ℃ to grow and isolate the bacteria. The search for antagonistic microorganisms was selected from microorganisms capable of growing even on antibiotics with inhibitory activity produced by root rot pathogens. Searching for antagonistic microorganisms primarily inhibits the growth of Fusarium Solani strains by first separating the selected microorganisms and spreading the strains to be searched during the cultivation of Fusarium Solani strains, the root rot-causing bacteria, on agar medium per starch. Antagonistic microorganisms with this were selected secondarily.

Primary screened Bacillus subtilis (B. subtilis) Was treated with caffein at a concentration of 50 mM, and the surviving strains were again exposed to ultraviolet light to prevent the mutation-induced antagonistic strains.Cylindrocarpon destructans) Strains showing inhibitory activity to the strains were selected (FIG. 1). Bacillus subtilis, which had inhibitory activity on the Syringrocapon destructhans strain, was inoculated with LBG (tryptone 1.0%, yeast extract 0.5%, NaCl 1.0%, glucose 1.0%), incubated for 12-18 hours, and 5 mM caffeine, 0.1 M MgSO₄7H₂O, 4 ml of bacterial solution containing 0.03% tween 80 (A₅₅₀= 0.4) was dispensed onto a glass plate. After dispensing, 2 short-wave UV lamps (10W) attached to the top of 20cm and one long-wave UV lamp were irradiated for 10 seconds to 2 minutes, and the surviving strains in conditions of 0.1% survival (90 seconds) were smeared onto the LB agar plate. After incubation at 30 ℃ strain was selected. Selected strains were cultured in LB liquid medium and then produced medium (sucrose 30g, soybean meal 20G, MnCl₂4H₂O 5mg, FeSO₄7H₂Strains with increased inhibitory activity compared to the parent strain were selected from the high titer test specimens of Syringrocarpon destructhans by incubating for 24 to 48 hours in O 50mg per liter).

Example 2 Identification of Antagonistic Microbes Exploded

The sequence of the gene (rDNA) encoding 16S rRNA of the antagonistic microbial strain selected in Example 1 is shown in Figure 2 (SEQ ID NO: 1). Compared with the rDNA sequences of known Bacillus subtilis strains AB017589 and AB017590, the rDNA sequences of Bacillus subtilis selected in Example 1 described in SEQ ID NO: 1 are known from Bacillus subtilis strains AB017589 and 99 % Similarity and 99% concordance with another known Bacillus subtilis strain AB017590.

After confirming the form of the Bacillus subtilis strain selected in Example 1 by an optical microscope and magnified at 8800 times magnification in an electron microscope, the size of the strain was 0.6-0.7 X 1.5-1.6㎛ on average The size of the general Bacillus subtilis was slightly smaller than 1.2-2.5 μm, with one flagella having a length of approximately 7.5 μm, which was about 5 times the size of the cell (FIG. 3).

However, if there are many strains that match 99% or more of the sequence similarity, it was judged that it was impossible to identify them. Therefore, Chun and Goodfellow method (Chun J. & Goodfellow M. (1995) Aphylogenetic analysis of the genus Nocardia with 16S rRNA gene were obtained by incubating the antagonistic microorganism strains at 37 ° C. for 24 hours in nutrient agar plate medium. The DNA was obtained according to Int. J. Syst. Bacteriol. 45: 240-245) and the gyrA gene was identified as p-gyrA-f (5'-CAG TCA GGA AAT GCG TAC GTC CTT-3 ', SEQ ID NO: 2). p-gyrA-r (5'-CAA GGT AAT GCT ATT GCT-3 ', SEQ ID NO: 3) PCR was performed using two kinds of primers to amplify the gene, and PCR conditions and sequencing were analyzed by Chun and Goodfellow methods. Accordingly. As a result, the new strains were classified into Bacillus subtilis based on the comparison data of 16S rDNA sequences of the microorganisms most used to identify the location of the new strains. Based on the characteristics of the strain selected in Example 1 of the present invention was named a new strain "Bacillus subtilis B-5604", on September 13, 2010 Korea Biotechnology Research Institute Biological Resource Center (Daejeon, Korea) It was deposited with Accession No. KCTC 11758BP in Yuseong-gu Hagyuro-ro 111).

Example 3: Culture conditions of Bacillus subtilis B-5604

Bacillus subtilis B-5604, an antagonistic microorganism with inhibitory activity against ginseng root rot, was cultured by shaking at 28 ° C for 24 hours in LB medium (1% casein peptone, 0.5% yeast extract, 0.5% salt, after autoclaving). And 20% glycerin were mixed at a ratio of 1: 1 and placed in frozen vials, each of which was used for culturing the active material while being frozen at -70 ° C.

To produce an active substance with inhibitory activity against ginseng root rot from activated microorganisms, the frozen Bacillus subtilis B-5604 vial is dissolved and the active substance production medium (1% soluble starch, 0.5% Pharmamade 0.5%) in a 1-liter Erlenmeyer flask. , Inoculated into 100 ml of glucose 0.2%, ammonium sulfate 0.1%, potassium phosphate 0.1%, magnesium sulfate 7 times crystallized water 0.05%, calcium chloride 0.1%, sodium chloride 0.3%, initial pH 7.0) and at 28 ° C. for 48 hours. Shake culture was performed at a speed of 200 rpm. The foam defoaming agent produced at the time of culture used an appropriate amount of silicone oil.

Example 4 Fusarium Solani or Cylindrocarpon Distractans Shown by Bacillus subtilis strain B-5604 Growth Inhibitory Effect of Strains

Bacillus subtilis (Bacillus subtilisStrain B-5604 is Fusarium Solani or Cylindrocarpon distractans To confirm the fact that it inhibits the growth of strains, Fusarium Solani or Syringrocarpon destructans in PDA medium The growth inhibition activity of the strain was analyzed. Fusarium solani or cyndrocarpon distractans incubated for 72 hours at 37 ° C. in PDA medium The strain was cultivated 3X3 mm with sterile spatula and placed on PDA medium, and Bacillus subtilis strain B-5604 was taken with sterile white gold teeth, side-by-side and incubated for 28 hours at 28 ° C. Leeum Solani or Cylindrocaphone Distantans It was confirmed that the growth of the strain is suppressed.

Example 5 Inhibitory Effect of Bacillus Subtilis Strain B-5604 on Ginseng Root Disease

In order to confirm the inhibitory activity of ginseng root rot on the active microorganisms, it was tested in farmland in Jeungpyeong, Chungcheongnam-do.

At the start of the test, the presence of root rot pathogens was confirmed in the ginseng arable land at the time of the test in May, and 2 years old ginseng was planted in 1 field out of 5000 (Note: the size of one cell is 1.8x 0.9 m and the ginseng used for the test) The number of ginseng roots was transplanted to 500 roots per year, and three quarters of the ginseng roots were covered with soil containing the root rot pathogen, and the same amount of vermiculite as the root rot control antagonism microorganism cultured by the method of Example 3 was placed thereon. Ginseng was harvested in August of the same year by covering the dried soil with adsorption on the back.

As a result of visual inspection of the improvement of root rot disease of ginseng against the active control of root rot disease, the soil part (lower part) where root rot pathogen was present was rotten as it was, but the drug adhered to the vermiculite etc. The part (upper part) which grew in the part was confirmed that the root rot disease was improved and the growth condition was good (FIG. 4).

In addition, the root rot inhibiting experiment of antagonistic microorganisms confirmed the presence of root rot pathogens in the ginseng farmland and transplanted 2 year old ginseng to 1 of 5000 ginseng ginseng planted in the cultivated land, and cultivated the control covered with soil containing root rot pathogens. . The test group transplanted 2 years old ginseng and cultivated the root rot disease control antagonist active microorganism cultured by the method of Example 3 to adsorb the same amount of vermiculite, and then mixed the dried vermiculite with soil present in the root rot pathogen 1: 1 In August of the same year, ginseng was harvested to investigate the improvement of root rot of ginseng against control and root rot control antagonist microorganisms.

As a result of visually determining the improvement of root rot disease of ginseng against the active microbial treatment of root rot, the soil containing root rot pathogen was rotted, but the vermiculite treated group adsorbed with antagonist active microorganism improved root rot disease. Was confirmed to be good (FIG. 5).

Claims (12)

1. Bacillus subtilis strain B-5604 (Accession Number: KCTC 11758BP).
2. Bacillus subtilis strain B-5604 (Accession No .: KCTC 11758BP) of claim 1 or a composition for the control of root rot pathogens comprising the culture medium thereof.
3. According to claim 2, wherein the root rot pathogen is a ginseng root rot pathogen is a composition for controlling the root rot pathogen.
4. The method of claim 3, wherein the ginseng root rot pathogen is cyrindrocarpon destructans (Cylindrocarpon destructans) or Fusarium Solani (Fusarium solaniA composition for controlling the root rot pathogen.
5. The composition of claim 3, wherein the composition is applied to ginseng root or the soil around it.
6. According to claim 2, wherein the composition is a rot rot pathogen control composition that is a hydrate, granules or capsules.
7. According to claim 2, wherein the composition is Bacillus subtilis strain B-5604 or a composition for the control of root rot pathogens that is prepared by adding a surfactant, a weight agent and a nutrient to the culture medium.
8. According to claim 2, wherein the effective amount of the microorganisms contained in the composition is 1 X 10 ⁶ to 1 X 10 ⁹ per ml (ml) composition for the control of root rot pathogens.
9. According to claim 2, wherein the effective amount of the microorganisms contained in the composition is 1 X 10 ⁷ to 5 X 10 ⁷ The number of microorganisms per arable land area (㎡) is sprayed composition for root rot pathogen control.
10. The method of controlling ginseng root rot pathogens using the composition of any one of Claims 2-9.
11. The method according to claim 10, wherein the composition is sprayed or immersed in the roots of the ginseng or the soil adjacent to the ginseng.
12. Cylindrocarpon distractans using Bacillus subtilis strain B-5604 (Accession No .: KCTC 11758BP) of claim 1 (Cylindrocarpon destructans) or Fusarium Solani (Fusarium solaniHow to suppress the growth of).

Images